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目的:探讨建立简便高效的大鼠海马神经元原代培养的纯化方法。方法:(1)离体培养新生24 h内SD乳鼠海马神经元,每天在倒置相差显微镜下进行神经元的形态学观察;(2)MTT法检测培养不同时间段神经元的活力变化,并绘制生长曲线;(3)纯化培养9 d神经元利用免疫荧光染色法检测β-微管蛋白Ⅲ(β-tublinⅢ)的表达率。结果:(1)倒置相差显微镜下观察原代培养的海马神经元,刚分离种植时可见神经细胞为圆形,24 h大多数细胞贴壁,3 d细胞突起增大、增粗,培养5~7 d神经元胞体丰满,突起交织成的网络更密集,培养14~16 d后,神经元开始退化;(2)生长曲线结果发现海马神经元体外培养经历了4个时期,即生长潜伏期、对数生长期、平台期、生长停滞期;(3)经β-tublinⅢ鉴定海马神经元纯度为(93.0%±2.5%)。结论:本实验方法步骤简便,可获得纯度较高的海马神经元,为研究与海马神经元相关的疾病提供了实验基础。
Objective: To explore a simple and efficient method of primary culture of hippocampal neurons in rats. Methods: (1) Hippocampal neurons of neonatal SD neonatal rats were cultured in vitro for 24 h. Morphological changes of neurons were observed under inverted phase contrast microscope. (2) MTT assay was used to detect the changes of neurons in different time periods. (3) Cultured 9-day neurons were used to detect the expression of β-tublinⅢ by immunofluorescence staining. Results: (1) Primary cultured hippocampal neurons were observed under an inverted phase contrast microscope. When the cells were isolated and planted, the nerve cells were round, and most of the cells adhered to the surface after 24 h. The protrusions of the cells grew and thickened on day 3, After 7 days, neurons became degenerated. After cultured for 14-16 days, the neurons began to degenerate. (2) The growth curve of hippocampal neurons cultured in vitro showed four stages, that is, the incubation period, (3) The purity of hippocampal neurons identified by β-tublin Ⅲ was (93.0% ± 2.5%). Conclusion: The experimental method is simple and easy to obtain high purity of hippocampal neurons, providing experimental basis for the study of neurons associated with hippocampal neurons.